Propionate is a three-carbon short-chain fatty acid that is very abundant in soil and the gastrointestinal tracts of animals, including humans. Propionate is widely used as a food preservative because it inhibits the growth of pathogenic and non-pathogenic prokaryotes and lower eukaryotes, but it does not affect human cells. The inhibitory effects of propionate have been attributed to the build up of the activated form of propionate, i.e., propionyl-coenzyme A (Pr-CoA). We have challenged this idea on the basis of published and unpublished data obtained from our group. In this application we propose to investigate the molecular basis of propionate toxicity. Our interpretation of the data suggests that growth arrest caused by propionate is the result of the synthesis of a non-physiological isomer of 2-methylcitrate by the citrate synthase (GltA) enzyme of the Krebs cycle;we refer to this inhibitor as 2-MCGltA. Our data suggest that 2-MCGltA inhibits an enzyme of gluconeogenesis. The lack of glucose has multiple negative effects. For example, cell wall and ribose syntheses are blocked, rendering the cell sensitive to osmotic changes and unable to make nucleic acids and coenzymes. One specific aim focuses on the identification and characterization of the target of 2-MCGltA. We seek to learn how 2-MCGltA inhibits the activity of the target, and whether the inhibitory effect of 2-MCGltA is stereospecific. A second specific aim focuses on the identification and analysis of ancillary functions needed to prevent propionate toxicity, and proteins involved in transporting propionate into the cell. PUBLIC HEALTH RELEVANCE: The project proposed in this application will provide insights into how propionate (a food preservative) exerts its inhibitory effects on cell growth at the molecular level. This work is relevant to human health and nutrition since propionate affects pathogenic and non-pathogenic microbes alike.